Single-stranded RNA viruses which are capable of replicating in the cytoplasm of host cells are widespread in nature. Those single-stranded viruses with message-sense genomic RNA molecules are called (+) strand, or positive strand, RNA viruses. Among the known (+) strand RNA viruses there are bacteria-specific, animal-specific, and plant-specific varieties. There is much diversity in the morphology of virus particles, coat proteins, genetic organization, and genome size. The (+) strand RNA viruses include, but are not limited to, Q-beta bacteriophage, poliovirus and alphaviruses (including Sindbis virus) of animal cells, and the bromoviruses (including brome mosaic virus) and the comoviruses (including cucumber mosaic virus) of plants.
A general review of (+) strand virus replication has been published (E. Strauss and J. Strauss (1983) Curr. Top. Microbiol. Immunol. 105:1). A brief summary of the steps follows. Virally determined proteins may be required together with the genomic RNA(S) for infection to occur, or all the viral proteins required to initiate virus replication may be synthesized using the incoming viral genomic RNA as message. There is no DNA intermediate in the replication of the nucleic acid of these viruses; the replication of the genetic information of a (+) strand RNA virus requires the RNA-dependent RNA synthetic capability of cells infected with such viruses. The incoming genomic (+) strand serves as template for the synthesis of (-) strand molecules, and then progeny (+) strands are synthesized using the (-) strands as templates. At the latter step there is an amplification of (+) strands over (-) strand molecules. The (+) strand viral RNA molecule(s) are generally translated, at least in part, to yield virus-specific replicase. The (-) strands serve as the templates for subsequent synthesis of large numbers of (+) strands which may carry structural genes and which are destined to become encapsidated by coat protein. Structural proteins are translated from genomic RNA or from subgenomic RNA, depending on the virus. In the instance of the (+) strand viruses with multicomponent RNA genomes, the RNA molecules may be encapsidated in separate viral particles, in which case the host cell must be simultaneously infected with each component to yield a productive infection.
The plant (+) strand RNA viruses have been classified into two supergroups (R. Goldbach (1986) Ann. Rev. Phytopathol. 24:289). The picornavirus-like supergroup includes comoviruses, nepoviruses, and potyviruses, while the second supergroup includes those viruses which resemble the animal alphavirus Sindbis virus: the tobamoviruses, tobraviruses, bromoviruses, cucumoviruses, and alfalfa mosaic virus. Although the (+) strand RNA viruses of plants and animals are diverse with respect to host range, genome and particle structures, and exact mechanisms of viral replication, there are some amino acid and nucleic acid sequence homologies (reviewed in R. Goldbach (1987) Microbiol. Sci. 4:197). Amino acid sequence homologies have been described for poliovirus, foot-and-mouth disease and cowpea mosaic viruses (H. Franssen et. al. (1984) EMBO J. 3:855), and for nonstructural proteins encoded by brome mosaic virus, alfalfa mosaic virus, tobacco mosaic virus and Sindbis virus (J. Haseloff et al. (1984) Proc. Nat. Acad. Sci. USA 81:4358; P. Ahlquist et al. (1985) J. Virol. 53:536). The homologous regions within the nonstructural proteins are believed to reflect mechanistic similarities in the replication of viral nucleic acid and possibly evolutionary relationships.
The alphaviruses and the Sindbis-like plant viruses also share a common strategy for synthesis of coat protein (and in some cases for the synthesis of additional protein(s). That strategy is the use of an internal promoter to direct the synthesis of a (+) strand RNA molecule, called a subgenomic RNA, using the (-) strand as template. The subgenomic RNA comprises a subset of sequences found in the corresponding "genomic" (full-length) RNA, and may be encapsidated in viral particles. In the case of the brome mosaic virus (BMV), (+) strand RNA molecules outnumber the full-length (-) strand template about 100 to 1, and subgenomic and full-length genomic RNAs are made in approximately equimolar amounts (French and Ahlquist (1987) J. Virol. 61:1457). The subgenomic mRNA, which in BMV carries only the message for coat protein, is very effectively translated by the cellular protein synthetic machinery. For example, gram quantities of BMV-infected plant tissue synthesize milligram amounts of coat protein (L. Lane (1981) in Handbook of Plant Virus Infections and Comparative Diagnosis, E. Kurstak, Ed., Elsevier/North-Holland, Amsterdam, pp. 333-376).
The bromoviruses are a group of (+) strand plant viruses with tripartite genomes. Included in this grouping are brome mosaic virus, cowpea chlorotic mosaic virus (CCMV), and broadbean mosaic virus (BBMV). CCMV and BBMV infect the dicots cowpeas and broadbeans respectively, and the host range of BMV is the grasses, including the cereals (L. C. Lane, (1981) in The Handbook of Plant Virus Infections and Comparative Diagnosis, Chapter 12, Elsevier, Amsterdam). Barley is a common experimental host for BMV, which has been well characterized.
BMV strain M1 (Madison 1) (P. Ahlquist et al. (1984) Proc. Nat. Acad. Sci. USA 81:7066) is the experimental system chosen to exemplify the present invention. The BMV genome is composed of three RNA molecules of unique, known sequence: RNA1 is 3234 bases in length, RNA2 is 2865 bases in length (P. Ahlquist et al. (1984) J. Mol. Biol. 172:369), and RNA3 is about 2114 bases in length (P. Ahlquist et al. (1981) J. Mol. Biol. 153:23). Complete cDNA clones have been produced (P. Ahlquist and M. Janda (1984) Mol. Cell. Biol. 4:2876). RNAs 1 and 2 are individually encapsidated; RNA3 is encapsidated together with RNA4, the 876 base subgenomic coat protein message which is presumed not to replicate in vivo (T. Lane (1974) Adv. Virus Res. 19:151). RNA molecules purified from virus particles and transcripts prepared from cloned viral cDNA sequences are infectious in the barley protoplast model system (P. Ahlquist et al. (1984) Proc. Nat. Acad. Sci. USA 81:7066). Therefore the full range of molecular biological techniques is available for the analysis of BMV nucleic acid functional sequences.
Experimental manipulations of the cloned BMV genome have begun to define regions of functional importance (P. Ahlquist and R. French (1988) in Domingo, Holland and Ahlquist (eds.) RNA Genetics Book 2: RNA Variability, chapter 3, CRC Press, Orlando, Fla.). Both RNAs 1 and 2 and their gene products are required for viral RNA synthesis; RNAs1 and 2 encode nonstructural proteins 1a and 2a respectively, and these proteins determine RNA-dependent RNA polymerase activity. RNA3 encodes nonstructural protein 3a and coat protein, but neither RNA3, protein 3a, nor coat protein is required for viral RNA replication (R. French and P. Ahlquist (1986) Science 231:1294). RNA3 can be modified by the insertion of a foreign gene in place of the coat protein gene, or by certain deletions, without the loss of replication ability (U.S. patent application Ser. No. 709,181, filed Mar. 7, 1985; R. French et al. (1986) Science 231:1294). RNA3 has been well studied because its gene products are not required for RNA replication (French et al. 1986). There are regions at the 5' and the 3' noncoding ends of the molecule which have homologs on RNAs 1 and 2, and it has been proposed that these sequences may function in viral RNA polymerase recognition, initiation of encapsidation, protection of the molecules from cellular nucleases, or a combination of these functions. Deletion studies using cloned cDNA and in vitro transcripts have shown that portions of the 5' and the 3' ends are required for genomic RNA replication, and that there is a graded effect of deletions in the intercistronic region of RNA3 as well (R. French and P. Ahlquist (1987) J. Virol. 61:1457). One feature of the intercistronic region is an oligo(A) tract, ranging from 16-22 nucleotides in length in natural virus populations (P. Ahlquist et al. (1981) J. Mol. Biol. 153:23). The subgenomic promoter of BMV is located in the intercistronic region as well. Neither the genomic nor the subgenomic promoters of the BMV are recognized by host RNA polymerases; only virally infected cells have RNA-dependent RNA polymerase activity capable of responding to these signals.
W. Miller et al., 1985, Nature 313:68, established that the production of RNA4, which serves as the message for coat protein, occurs by initiation of RNA synthesis within the RNA3 (-) strand molecule. (-) strand RNA3 templates ending at the BqlII site about 21 bases upstream of the RNA4 initiation site were able to direct RNA4 production (Miller et al. (1985); L. Marsh et al. (1987) in Positive Strand RNA Viruses, New York, Alan R. Liss, Inc., pp. 327-336. These studies and a study by R. French et al. (1986) Science 231:1294, show that deletions and insertions at or downstream of the SalI site, which is about 17 bases downstream of the start site, do not interfere with subgenomic mRNA production. French et al, 1986, demonstrated the expression of suitably inserted foreign genes by the subgenomic pathway. Thus, BMV subgenomic promoter activity was thought to be localized to a 37 base region which in the cDNA clone lies between the BglII and the SalI sites. Marsh et al. (1987) also presented evidence from in vitro experiments that the 5' boundary of the BMV subgenomic promoter activity was not further upstream than the BglII site about 21 bases upstream of the RNA4 start site. Marsh et al. (1987) also stated, without evidence, that the poly(A) tract somewhat further upstream of the RNA4 initiation site improved promoter activity. Both the studies by Miller et al. (1985) and those by Marsh et al. (1987) utilized in vitro expression experiments to examine the requirements of transcription; their results did not indicate that nucleotide sequence information as far upstream as -95 could contribute to the level of (+) strand mRNA synthesis. Neither Miller et al. (1985) nor Marsh et al. (1987) reported activity of subgenomic transcription, since in their constructs, sequences 5' to the putative promoter were missing. No published study has described the use of a subgenomic promoter-containing fragment of nucleic acid which could be transferred to a desired new location to generate a novel subgenomic mRNA.